Production of fatty acids esters

ABSTRACT

A microbial cell is used for producing at least one fatty acid ester, wherein the cell is genetically modified to contain (i) at least one first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A (CoA) thereof by increased enzymatic activity in the cell relative to the wild type cell of malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway, wherein the fatty acid contains at least 5 carbon atoms; and (ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell and the wax ester synthase has sequence identity of at least 50% to a polypeptide of SEQ ID NO: 1-8 and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the fatty acid ester.

This application is a continuation of U.S. application Ser. No.14/843,525, filed Sep. 2, 2015, and entitled PRODUCTION OF FATTY ACIDSESTERS, which claims the benefit of U.S. Provisional Application No.62/044,621, filed Sep. 2, 2014, and entitled PRODUCTION OF FATTY ACIDSESTERS, each of which is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a biotechnological method and cell forproducing at least one fatty acid ester from a sugar.

Discussion of the Background

Fatty acid esters may be used for several purposes commercially. Forexample, biodiesel, an alternative fuel, is comprised of esters (e.g.,fatty acid methyl ester, fatty acid ethyl esters, etc.). Some lowmolecular weight esters are volatile with a pleasant odour which makesthem useful as fragrances or flavouring agents. Fatty acid esters mayalso be used as solvents for lacquers, paints, varnishes and the like.Esters are also used as softening agents in resins and plastics,plasticizers, flame retardants, and additives in gasoline and oil.Further, esters can be used in the manufacture of polymers, films,textiles, dyes, and pharmaceuticals. Accordingly, fatty acid esters arevery useful in this day and age.

Fatty acid esters may be extracted from petroleum. However, this methodis energy consuming and costly. Also, it is an inefficient processbecause frequently the long chain hydrocarbons in crude petroleum arecracked to produce smaller monomers. These monomers are then used as theraw material to manufacture the more complex specialty chemicals. Thisprocess of cracking gasoline or petroleum is bad for the environment.Also, since the costs for these starting materials will be linked to theprice of petroleum, with the expected increase in petroleum prices inthe future, cost of making these fatty acid esters may also increaserelative to the increase in the petroleum prices.

Due to the inherent challenges posed by petroleum, there is a need for arenewable petroleum source which does not need to be explored,extracted, transported over long distances, or substantially refinedlike petroleum. There is also a need for a renewable petroleum sourcethat can be produced economically and that does not cause theenvironmental damage as that produced by the petroleum industry and theburning of petroleum based fuels.

Fatty acid esters may be found in several biofuels. However, the yieldof the fatty acid esters from these biofuels and/or plant based fuels islow. Thus, a need exists to develop an alternate biological source offatty acid esters. One option is to recombinantly engineer a microbialspecies for efficient production of fatty acid esters.

Fatty acid esters are known to be the product of a condensation reactionbetween an acyl-CoA molecule and an alcohol of any chain lengthsometimes in the presence of wax ester synthases. For example, a fattyacid ester can be the condensation product of methanol, ethanol,propanol, butanol, isobutanol, 2-methylbutanol, 3-methylbutanol, orpentanol with an acyl-CoA molecule. In some instances, fatty acid esterssuch as fatty acid methyl esters (“FAME”) or fatty acid ethyl esters(“FAEE”) can be produced by supplying the alcohol used in the reaction(e.g., methanol or ethanol) to the culture media. Similarly, wax esterscan be produced by supplying fatty alcohols.

Most fatty acid esters have useful functions as mentioned above. One ofthese esters, methyl laurate, CH₃(CH₂)₁₀COOCH₃ a water-insoluble, clear,colourless ester, has several uses in the commercial industry includingthe pharmaceutical and cosmetic industry.

However, the current methods used to make fatty acid esters areinefficient as they produce a large amount of by-products that result ina waste of resources. Also, the currently available methods do not allowfor selecting specific fatty acid esters. There is thus a need for moreenergy efficient and specific production of fatty acid esters includingmethyl laurate.

SUMMARY OF THE INVENTION

The present invention attempts to solve the problems above by providingat least one method of producing fatty acid esters from geneticallyengineered microorganisms. In particular, the fatty acid esters areproduced by culturing a microorganism that is genetically engineered toproduce a fatty acid and express at least one wax ester synthase, in thepresence of exogenous alcohol, such as exogenous ethanol, exogenousmethanol or the like.

In one embodiment, the present invention relates to a microbial cell forproducing at least one fatty acid ester, wherein the cell is geneticallymodified to comprise

-   -   (i) at least one first genetic mutation that enables the cell to        produce at least one fatty acid and/or acyl coenzyme A (CoA)        thereof by increased enzymatic activity in the cell relative to        the wild type cell of malonyl-CoA dependent and malonyl-ACP        independent fatty acyl-CoA metabolic pathway, wherein the fatty        acid comprises at least 5 carbon atoms; and    -   (ii) a second genetic mutation that increases the activity of at        least one wax ester synthase in the cell relative to the wild        type cell and the wax ester synthase has sequence identity of at        least 50% to a polypeptide of SEQ ID NO: 1-8 and combinations        thereof or to a functional fragment of any of the polypeptides        for catalyzing the conversion of fatty acid and/or acyl coenzyme        A thereof to the fatty acid ester.

In another embodiment, the present invention relates to a method forproducing methyl laurate, the method comprising:

contacting lauric acid and/or lauroyl coenzyme A with an isolated waxester synthase that has sequence identity of at least 50% to apolypeptide of SEQ ID NOs: 1-8 and combinations thereof.

The present invention also related to the above method, which is carriedout within a microbial cell which is genetically modified to comprise

-   -   (i) at least one first genetic mutation that enables the cell to        produce at least one fatty acid and/or acyl coenzyme A (CoA)        thereof by increased enzymatic activity in the cell relative to        the wild type cell of malonyl-CoA dependent and malonyl-ACP        independent fatty acyl-CoA metabolic pathway, wherein the fatty        acid comprises at least 5 carbon atoms; and    -   (ii) a second genetic mutation that increases the activity of at        least one wax ester synthase in the cell relative to the wild        type cell and the wax ester synthase has sequence identity of at        least 50% to a polypeptide of SEQ ID NO: 1-8 and combinations        thereof or to a functional fragment of any of the polypeptides        for catalyzing the conversion of fatty acid and/or acyl coenzyme        A thereof to the fatty acid ester.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing free fatty acid (FFA) production in shakeflask cultures after 68 hours for four FFA production strains.

FIG. 2 is a graph showing the lauric acid production profile for BXF_169evaluated in 50 ml shake flask, OD₆₀₀=2.

FIG. 3 is a graph showing the mRNA expression relative to cysG forvarious pathway genes in BXF_169 measured at 8, 24 and 48 hrs.

FIG. 4 is a graph showing the enzyme activity for various elongationsteps measured in lysate for BXF_169 at 8 and 24 hours. EnCR activity isencoded by the Ter gene. KAS activities are encoded by the NphT7 andFabH genes.

FIG. 5 is a graph showing intermediate product formation and utilizationspecific to the C4→C6 elongation steps of the lauroyl-CoA productionpathway (data collected but not shown for the C6→C12 intermediates) fromin vitro pathway reconstitution with FadB, FadB+Crt and FadB+Crt+Hbd.

FIG. 6 is a graph showing product formation measured for 20 soluble WEScandidates. Purified candidate enzymes were incubated individually withmethanol and C6- to C14-CoA substrates to assess product formation.

FIG. 7 is a graph showing FAME production at 24 hours for ninethiolase-based strains expressing different wax ester synthasecandidates evaluated with the 2 mL small-scale protocol.

FIG. 8 is a graph showing the production of methyl laurate and otherFAMEs (C6-C14) in 1 ml, 24 hour strain screening assay by 50 strainsengineered with various versions of the synthase pathway for methyllaurate production.

FIG. 9 is a graph showing FAME product distribution exhibited by strainBXE_022 after 40 hrs in 1 L fermentation under various test conditions.

FIG. 10 is a graph showing FAME and FFA concentration distribution forstrains BXE_058 and BXE_062 at approximately 45 hours of production;methyl myristate concentration was not include in the analysis as 50 g/Lwas added as a second phase.

FIG. 11 is an illustration of the metabolic pathways of a cell relatedto genetic modifications for increasing flux through the intermediatemalonyl-CoA

FIG. 12 is a graph showing the evaluation of intermediate productaccumulation specific to the C4→C6 elongation steps of the lauroyl-CoAproduction pathway (data collected but not shown for the C6→C12intermediates.)

FIG. 13 is a graph showing specific enzyme activities for three FabHmutant enzymes with C6-C10-CoA substrates compared to wild-type FabH.

FIG. 14 is a graph showing FAME production in standard 1 mL method after20 hours of production. (Note: BXE_198 is the control comparison forBXE_271-273 and BXE_233 is the control for BXE_243-244).

FIG. 15 is a graph showing FAME production in standard 1 mL method after20 hours of production with alternative WES enzyme candidates.

FIG. 16 is a graph showing in vitro product formation from methanol andC12-CoA after incubation for 5 minutes with purified Mhyd variants asmeasured by GC-MS.

FIG. 17 is a graph showing FAME production in standard 1 mL method after20 hours of production with Maqu mutants (SEQ ID NOs:7 and 8).

FIG. 18 is a graph showing WES enzyme activities for BXE_062 and BXE_233cell lysates as measured by product formation over time by GC-MS. Thetarget activity for WES in cell lysates is 0.02 U/mg.

FIG. 19 is a graph showing average production profile for methyl laurateand total FAME for 1 L fermentations with BXE_276 in the FM14 process.

DETAILED DESCRIPTION OF THE INVENTION

The ranges described below include all values and subvalues between thelower and upper limit of the range.

According to one aspect, the present invention provides a microbial cellfor producing at least one fatty acid ester, wherein the cell isgenetically modified to comprise:

-   -   at least one first genetic mutation that enables the cell to        produce at least one fatty acid and/or acyl coenzyme A thereof,        wherein the fatty acid comprises at least 5 carbon atoms; and    -   at least one second genetic mutation that increases the activity        of at least one wax ester synthase in the cell relative to the        wild type cell and the wax ester synthase has sequence identity        of at least 50% to a polypeptide of SEQ ID NOs: 1-8 and        combinations thereof or to a functional fragment of any of the        polypeptides for catalyzing the conversion of fatty acid and/or        acyl coenzyme A thereof to the fatty acid ester.

In particular, the cell may be capable of producing the fatty acidand/or acyl coenzyme A thereof by means of increased enzymatic activityin the cell relative to the wild type cell of the malonyl-CoA dependentand malonyl-ACP independent fatty acyl-CoA metabolic pathway.

The microbial cells according to any aspect of the present invention maybe prokaryotes or eukaryotes. These can be mammalian cells (such as, forexample, cells from man), plant cells or microorganisms such as yeasts,fungi or bacteria, wherein microorganisms in particular bacteria andyeasts may be used.

Suitable bacteria, yeasts or fungi are in particular those bacteria,yeasts or fungi that are deposited in the Deutsche Sammlung vonMikroorganismen and Zellkulturen (German Collection of Microorganismsand Cell Cultures) GmbH (DSMZ), Brunswick, Germany, as bacterial, yeastor fungal strains. Bacteria suitable according to the invention belongto the genera that are listed in the Deutsche Sammlung vonMikroorganismen and Zellkulturen (German Collection of Microorganismsand Cell Cultures) GmbH(DSMZ) Germany.

Yeasts suitable according to the invention belong to the genera that arelisted in the Deutsche Sammlung von Mikroorganismen and Zellkulturen(German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ),Brunswick, Germany.

Fungi suitable according to the invention belong to the genera that arelisted in the Deutsche Sammlung von Mikroorganismen and Zellkulturen(German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ),Brunswick, Germany.

In particular, the cells may be selected from the genera Aspergillus,Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes,Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula,Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia,Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter,Burkholderia, Clostridium and Cupriavidus. More in particular, the cellsmay be selected from the group consisting of Aspergillus nidulans,Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillussubtilis, Brevibacterium flavum, Brevibacterium lactofermentum,Burkholderia andropogonis, B. brasilensis, B. caledonica, B. caribensis,B. caryophylli, B. fungorum, B. gladioli, B. glathei, B. glumae, B.graminis, B. hospita, B. kururiensis, B. phenazinium, B. phymatum, B.phytofirmans, B. plantarii, B. sacchari, B. singaporensis, B.sordidicola, B. terricola, B. tropica, B. tuberum, B. ubonensis, B.unamae, B. xenovorans, B. anthina, B. pyrrocinia, B. thailandensis,Candida blankii, Candida rugosa, Corynebacterium glutamicum,Corynebacterium efficiens, Escherichia coli, Hansenula polymorpha,Kluveromyces lactis, Methylobacterium extorquens, Paracoccus versutus,Pseudomonas argentinensis, P. borbori, P. citronellolis, P. flavescens,P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P.resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P.chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P.azotoformans, ‘P. blatchfordae’, P. brassicacearum, P. brenneri, P.cedrina, P. corrugata, P. fluorescens, P. gessardii, P. libanensis, P.mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P.mucidolens, P. orientalis, P. panacis, P. proteolytica, P. rhodesiae, P.synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans,P. pertucinogena, P. cremoricolorata, P. fulva, P. monteilii, P.mosselii, P. parafulva, P. putida, P. balearica, P. stutzeri, P.amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens,P. ficuserectae, ‘P. helianthi’, P. meliae, P. savastanoi, P. syringae,P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici,P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P.azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii,P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P.frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P.indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P.koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P.pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P.poae, P. pohangensis, P. psychrophila, P. psychrotolerans, P. rathonis,P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P.salomonii, P. segitis, P. septica, P. simiae, P. suis, P.thermotolerans, P. aeruginosa, P. tremae, P. trivialis, P. turbinellae,P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P.xanthomarina, Ralstonia eutropha, Rhodospirillum rubrum, Rhodobactersphaeroides, Saccharomyces cerevisiae, Yarrowia lipolytica and Zymomonasmobile. More in particular, the cell may be a bacterial cell selectedfrom the group consisting of Pseudomonas, Corynebacterium, Bacillus andEscherichia. Even more in particular, the cells may be selected from thegroup consisting of Pseudomonas putida and Escherichia coli.

The genetically modified cell may be genetically different from the wildtype cell. The genetic difference between the genetically modified cellaccording to any aspect of the present invention and the wild type cellmay be in the presence of a complete gene, amino acid, nucleotide etc.in the genetically modified cell that may be absent in the wild typecell. In one example, the genetically modified cell according to anyaspect of the present invention may comprise enzymes that enable thecell to produce at least one fatty acid and/or acyl coenzyme A thereof;and convert the fatty acid and/or acyl coenzyme A thereof to the fattyacid ester. The wild type cell relative to the genetically modified cellof the present invention may have none or no detectable activity of theenzymes that enable the genetically modified cell to produce at leastone fatty acid and/or acyl coenzyme A thereof; and the enzymes thatenable genetically modified cell to convert the fatty acid and/or acylcoenzyme A thereof to the respective fatty acid ester.

The phrase “wild type” as used herein in conjunction with a cell maydenote a cell with a genome make-up that is in a form as seen naturallyin the wild. The term may be applicable for both the whole cell and forindividual genes. The term “wild type” therefore does not include suchcells or such genes where the gene sequences have been altered at leastpartially by man using recombinant methods.

A skilled person would be able to use any method known in the art togenetically modify a cell. According to any aspect of the presentinvention, the genetically modified cell may be genetically modified sothat in a defined time interval, within 2 hours, in particular within 8hours or 24 hours, it forms at least twice, especially at least 10times, at least 100 times, at least 1000 times or at least 10000 timesmore fatty acid and/or acyl coenzyme A thereof and the respective fattyacid ester than the wild-type cell. The increase in product formationcan be determined for example by cultivating the cell according to anyaspect of the present invention and the wild-type cell each separatelyunder the same conditions (same cell density, same nutrient medium, sameculture conditions) for a specified time interval in a suitable nutrientmedium and then determining the amount of target product (fatty acid,acyl coenzyme A thereof and the respective fatty acid ester) in thenutrient medium.

In particular, the cell comprises at least one first genetic mutationthat enables the cell to produce at least one fatty acid and/or acylcoenzyme A thereof. In particular, the first genetic mutation may enablethe cell to produce at least one fatty acid and/or acyl coenzyme Athereof by means of a malonyl-CoA dependent and malonyl-ACP independentfatty acyl-CoA metabolic pathway. More in particular, there is anincrease in enzymatic activity in the malonyl-CoA dependent andmalonyl-ACP independent fatty acyl-CoA metabolic pathway in the cellrelative to the wild type cell.

The cell may be genetically modified for increased enzymatic activity inthe microorganism's malonyl-CoA dependent, malonyl-ACP independent,fatty acyl-CoA metabolic pathway (“MDMIFAA”) This pathway is alsoreferred to herein as malonyl-CoA dependent, but malonyl-ACPindependent, fatty acyl-CoA metabolic pathway. Such increase in thecell's malonyl-CoA dependent, malonyl-ACP independent fatty acyl-CoAmetabolic pathway can be achieved by an increased activity or expressionof a gene or a pathway comprising an acetoacetyl-CoA synthase, aketoacyl-CoA synthase (or elongase), an enoyl-CoA reductase, aketoacyl-CoA reductase and/or a 3-hydroxyacyl-CoA dehydratase incombination with a decrease in expression or activity of acetoacetyl-CoAthiolase. Alternatively, increased activity in the microorganism'smalonyl-CoA dependent, malonyl-ACP independent fatty acyl-CoA metabolicpathway can be achieved by an increased expression of a gene or apathway comprising an acetoacetyl-CoA synthase, a ketoacyl-CoA thiolase,a enoyl-CoA reductase, a ketoacyl-CoA reductase and/or a3-hydroxyacyl-CoA dehydratase in combination with a decrease inexpression or activity of acetoacetyl-CoA thiolase.

A list of non-limiting genetic modifications to enzymes or enzymaticactivities that may lead a cell to produce a fatty acid and/or acylcoenzyme A thereof and that may be considered as the first geneticmutation according to any aspect of the present invention are providedbelow in Table 1 and explained in US20140051136.

In particular, fatty acid biosynthetic pathways in the cells of thepresent invention use precursors acetyl-CoA and malonyl-CoA. The enzymesthat may be involved are provided in FIG. 11.

In one example, nucleic acid sequences that encode temperature-sensitiveforms of these polypeptides may be introduced in place of the nativeenzymes, and when such genetically modified microorganisms are culturedat elevated temperatures (at which these thermolabile polypeptidesbecome inactivated, partially or completely, due to alterations inprotein structure or complete denaturation), there is observed anincrease in a chemical product. For example, in E. coli, thesetemperature-sensitive mutant genes could include FabIts(S241F),FabBts(A329V) or FabDts(W257Q) amongst others. In most of theseexamples, the genetic modifications may increase malonyl-CoA utilizationso that there is a reduced conversion of malonyl-CoA to fatty acids viathe native pathway, overall biomass, and proportionally greaterconversion of carbon source to a chemical product including a fatty acidor fatty acid derived product via a malonyl-CoA dependent andmalonyl-ACP independent route. Also, additional genetic modifications,such as to increase malonyl-CoA production, may be made for someexamples.

In another example, the enzyme, enoyl-acyl carrier protein reductase (ECNo. 1.3.1.9, also referred to as enoyl-ACP reductase) is a key enzymefor fatty acid biosynthesis from malonyl-CoA. In Escherichia coli thisenzyme, FabI, is encoded by the gene FabI (Richard J. Heath et al.,1995).

In one example, the expression levels of a pyruvate oxidase gene (Changet al., 1983, Abdel-Ahmid et al., 2001) can be reduced or functionallydeleted in the cell according to any aspect of the present invention.The pyruvate oxidase gene may encode an enzyme of, for example, EC1.2.3.3. In particular, the pyruvate oxidase gene may be a PoxB gene.

In one example, the expression levels of a lactate dehydrogenase gene(Mat-Jan et al., Bunch et al., 1997) can be reduced or functionallydeleted. In some examples, the lactate dehydrogenase gene encodes anenzyme of, for example, EC 1.1.1.27. The lactate dehydrogenase gene maybe an NAD-linked fermentative D-lactate dehydrogenase gene. Inparticular, the lactate dehydrogenase gene is an ldhA gene.

In one example, the first genetic mutation may be in at least onefeedback resistant enzyme of the cell that results in increasedexpression of the feedback resistant enzyme. In particular, the enzymemay be pantothenate kinase, pyruvate dehydrogenase or the like. In E.coli, these feedback resistant mutant genes could include CoaA(R106A)and/or 1pd(E354K).

In a further example, the increase in the cell's malonyl-CoA dependent,but malonyl-ACP independent fatty acyl-CoA metabolic pathway may occurthrough reduction in the acetoacetyl-CoA thiolase activity and/ortrigger factor activity and/or in the activity of a molecular chaperoneinvolved in cell division. In one example, the cell may comprise agenetic mutation in Ttig gene.

In one example, the first genetic mutation in the cell may result inincreased enzymatic activity in the NADPH-dependent transhydrogenasepathway relative to the wild type cell. This result may occur byintroduction of a heterologous nucleic acid sequence coding for apolypeptide encoding nucleotide transhydrogenase activity.

In another example, the first genetic mutation in the cell may result indecreased expression of fatty acyl-CoA synthetase and/or ligase activityvia any method known in the art.

In yet another example, the first genetic mutation in the cell mayresult in overexpression of an enzyme having acetyl-CoA carboxylaseactivity.

In one example, the cell may have increased intracellular bicarbonatelevels brought about by introduction of a heterologous nucleic acidsequence coding for a polypeptide having cyanase and/or carbonicanhydrase activity.

More in particular, the first genetic mutation according to any aspectof the cell may result in increased and/or decreased levels of fattyacyl-CoA thioesterase activity. This result may increase chain lengthspecificity of a desired fatty acid product by increasing levels ofchain length specific fatty acyl-CoA thioesterase activity anddecreasing the activity of fatty acyl-CoA thioesterase activity onundesired fatty acid chain lengths. In one example, the increased chainlength specificity of fatty acid or fatty acid derived product may occurby increasing levels of chain length specific ketoacyl-CoA thiolase,enoyl-CoA reductase, ketoacyl-CoA reductase or 3-hydroxyacyl-CoAdehydratase activities either individually or in combination.

The first genetic mutation in the cell according to any aspect of thepresent invention may result in an increase or decrease in expression ofonly one enzyme selected from the list of enzymes mentioned above or anincrease or decrease in expression of a combination of enzymes mentionedabove.

In another example, the first genetic mutation in the cell may be in atleast one enzyme selected from the group consisting of acetoacetyl-CoAsynthase, ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase,ketoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase andacetoacetyl-CoA thiolase. More in particular, the first genetic mutationin the cell may result in an increase in expression of acetoacetyl-CoAsynthase, ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase,ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase in combinationoptionally with a decrease in expression or activity of acetoacetyl-CoAthiolase. In particular, the enoyl-CoA reductase and/or ketoacyl-CoAreductase may either utilize the cofactor NADH and/or NADPH. Inparticular, the genetic modification in the cell according to any aspectof the present invention may comprise any of the enzymes listed in Table1 in combination with the following enzymes acetoacetyl-CoA synthase,ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase, ketoacyl-CoAreductase and/or 3-hydroxyacyl-CoA dehydratase and acetoacetyl-CoAthiolase wherein the expression or activity of enzymes acetoacetyl-CoAsynthase, ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase,ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase is increasedand the activity of acetoacetyl-CoA thiolase is decreased.

In yet another example, malonyl-CoA dependent, malonyl-ACP independentfatty acyl-CoA metabolic pathway in the cell according to any aspect ofthe present invention can be achieved by an increased expression of agene or a pathway comprising acetoacetyl-CoA synthase, ketoacyl-CoAthiolase, enoyl-CoA reductase, ketoacyl-CoA reductase and/or3-hydroxyacyl-CoA dehydratase in combination with a decrease inexpression or activity of acetoacetyl-CoA thiolase.

In particular, the first genetic modification in the cell according toany aspect of the present invention may comprise any of the enzymeslisted in Table 1 in combination with the following enzymesacetoacetyl-CoA synthase, ketoacyl-CoA thiolase, enoyl-CoA reductase,ketoacyl-CoA reductase and/or 3-hydroxyacyl-CoA dehydratase incombination with a decrease in expression or activity of acetoacetyl-CoAthiolase.

In one example, the cell according to any aspect of the presentinvention may comprise a first genetic modification in any of theenzymes listed in Table 1 in combination with the following enzymesacetyl-CoA carboxylase, malonyl-CoA:ACP transacylase (FabD),β-ketoacyl-ACP synthase III, β-ketoacyl-ACP synthase I (FabB),β-ketoacyl-ACP synthase II (FabF), 3-oxoacyl-ACP-synthase I and enoylACP reductase.

More in particular, the first genetic mutation may result in an increasein the expression of at least one enzyme selected from the groupconsisting of acetyl-CoA carboxylase, malonyl-CoA:ACP transacylase(FabD), β-ketoacyl-ACP synthase III, β-ketoacyl-ACP synthase I (FabB),β-ketoacyl-ACP synthase II (FabF), 3-oxoacyl-ACP-synthase I and enoylACP reductase relative to the wild type cell. In particular, the firstgenetic mutation may result in an increase in the expression of morethan one enzyme in the cell according to any aspect of the presentinvention that enables the cell to produce a fatty acid and/or acylcoenzyme A thereof by means of increased enzymatic activity in the cellrelative to the wild type cell of malonyl-CoA dependent and malonyl-ACPindependent fatty acyl-CoA metabolic pathway.

In one example, there may be an increase in expression of β-ketoacyl-ACPsynthase and 3-oxoacyl-ACP-synthase in the cell according to any aspectof the present invention. In another example, there may be an increasein expression of β-ketoacyl-ACP synthase and Malonyl-CoA-ACPtransacylase in the cell according to any aspect of the presentinvention. In yet another example, there may be an increase inexpression of β-ketoacyl-ACP synthase and enoyl ACP reductase in thecell according to any aspect of the present invention. In one example,there may be an increase in expression of β-ketoacyl-ACP synthase,Malonyl-CoA-ACP transacylase and enoyl ACP reductase in the cellaccording to any aspect of the present invention. In all examples, theremay be an increase in the expression of enoyl ACP reductase and/oracyl-CoA thioesterase.

The phrase “increased activity of an enzyme”, as used herein is to beunderstood as increased intracellular activity. Basically, an increasein enzymatic activity can be achieved by increasing the copy number ofthe gene sequence or gene sequences that code for the enzyme, using astrong promoter or employing a gene or allele that code for acorresponding enzyme with increased activity and optionally by combiningthese measures. Genetically modified cells used according to any aspectof the present invention are for example produced by transformation,transduction, conjugation or a combination of these methods with avector that contains the desired gene, an allele of this gene or partsthereof and a vector that makes expression of the gene possible.Heterologous expression is in particular achieved by integration of thegene or of the alleles in the chromosome of the cell or anextra-chromosomally replicating vector.

Accordingly, the cells and methods of the present invention may compriseproviding a genetically modified microorganism that comprises both aproduction pathway to a fatty acid or fatty acid derived product, and amodified polynucleotide that encodes an enzyme of the malonyl-ACPdependent fatty acid synthase system that exhibits reduced activity, sothat utilization of malonyl-CoA shifts toward the production pathwaycompared with a comparable (control) microorganism lacking suchmodifications. The methods involve producing the chemical product usinga population of such genetically modified microorganism in a vessel,provided with a nutrient media. Other genetic modifications describedherein, to other enzymes, such as acetyl-CoA carboxylase and/orNADPH-dependent transhydrogenase, may be present in some such examples.Providing additional copies of polynucleotides that encode polypeptidesexhibiting these enzymatic activities is shown to increase a fatty acidor fatty acid derived product production. Other ways to increase theserespective enzymatic activities is known in the art and may be appliedto various examples of the present invention.

TABLE 1 Examples of genetic modifications in cells of microorganisms forproduction of fatty acids and/or acyl coenzyme A thereof GeneticModifications E.C. CLASSIFICATION GENE NAME ENZYME FUNCTION No. IN E.COLI COMMENTS Glucose transporter N/A galP Increase function Pyruvatedehydrogenase E1p 1.2.4.1 aceE Increase function lipoateacetyltransferase/ 2.3.1.12 aceF Increase function dihydrolipoamideacetyltransferase Pyruvate dehydrogenase 1.8.1.4 lpd Increase functionor E3 (lipoamide dehydrogenase) alter such as by mutation to increaseresistance to NADH inhibition. Lactate dehydrogenase 1.1.1.28 ldhADecrease function, including by mutation Pyruvate formate lyase 2.3.1.—pflB Decrease function, (B “inactive”) including by mutation Pyruvateoxidase 1.2.2.2 poxB Decrease function, including by mutation Phosphateacetyltransferase 2.3.1.8 Pta Decrease function, including by mutationacetate kinase 2.7.2.15 2.7.2.1 ackA Decrease function, including bymutation methylglyoxal synthase 4.2.3.3 mgsA Decrease function,including by mutation Heat stable, histidyl N/A ptsH Decrease function,phosphorylatable protein (of PTS) (HPr) including by mutation Phosphoryltransfer protein N/A ptsI Decrease function, (of PTS) including bymutation Polypeptide chain (of PTS) N/A Crr Decrease function, includingby mutation 3-oxoacyl-ACP synthase I 2.3.1.179 fabF Decrease function,3-oxoacyl-ACP synthase II 2.3.1.41 including by mutation monomerβ-ketoacyl-ACP synthase I, 2.3.1.41 fabB Decrease function,3-oxoacyl-ACP-synthase I 2.3.1.— including by mutation Malonyl-CoA-ACP2.3.1.39 fabD Decrease function, transacylase including by mutationenoyl acyl carrier protein 1.3.1.9. fabI Decrease function, reductase1.3.1.10 including by mutation β-ketoacyl-acyl carrier 2.3.1.180 fabHDecrease function, protein synthase III including by mutation Carboxyltransferase 6.4.1.2 accA Increase function subunit α subunit Biotincarboxyl carrier 6.4.1.2 accB Increase function protein Biotincarboxylase subunit 6.3.4.14 accC Increase function Carboxyl transferase6.4.1.2 accD Increase function subunit β subunit long chain fatty acyl3.1.2.2. tesA Increase function as thioesterase I 3.1.1.5 well as alterby mutation to express in cytoplasm or deletion acyl-CoA synthase2.3.1.86 fadD Decrease via deletion or mutation acetate CoA-transferase2.8.3.8 atoD Decrease via deletion or mutation acetate CoA-transferase2.8.3.8 atoA Decrease via deletion or mutation Transporter N/A atoBDecrease via deletion or mutation acetyl-CoA acetyltransferase 2.3.1.9atoB Decrease via deletion or mutation pantothenate kinase 2.7.1.33 coaAIncrease via expression or feedback resisant mutation lactose repressorN/A lacI Decrease via deletion or mutation γ-glutamyl-γ- 1.2.1.— puuCDecrease via deletion aminobutymidehyrte or mutation dehydrogenasemalate synthase A 2.3.3.9 aceB Decrease via deletion or mutationisocitrate lyase 4.1.3.1 aceA Decrease via deletion or mutationisocitrate dehydrogenase 3.1.3.— aceK Decrease via deletionphosphatase/isocitrate 2.7.11.5. or mutation dehydrogenase kinasepyruvate formate-lyase 1.2.1.10 1.1.1.1 adhE Decrease via deletiondeactivate or mutation aldehyde dehydrogenase A, 1.2.1.21 1.2.1.22 aldADecrease via deletion NAD-linked or mutation acetaldehyde 1.2.1.4 aldBDecrease via deletion dehydrogenase or mutation Lambda phage DE3 lysogenN/A λDE3 Increase T7 mRNA polymerase N/A T7pol Increase trigger factor5.2.1.8 tig Decrease via deletion or mutation 3-ketoacyl-CoA thiolase2.3.1.16 fadA Increase dodecenoyl-CoA δ-isomerase, 5.3.3.8 1.1.1.35 fadBIncrease enoyl-CoA hydratase, 3- 5.1.2.3 4.2.1.17 hydroxybutyryl-CoAepimerase, 3-hydroxyacyl-CoA dehydrogenase Sucrose permease N/A cscBIncrease Invertase 3.2.1.26 cscA Increase fructokinase 2.7.1.4 cscKIncrease carbonic anhydrase 4.2.1.1 cynT Increase carbonic anhydrase4.2.1.1 can Increase pyridine nucleotide 1.6.1.2 pntAB Increasetranshydrogenase pyridine nucleotide 1.6.1.1 udhA Increasetranshydrogenase acyl-CoA thioesterase 3.1.2.20 3.1.2.2 yciA Increaseand or decrease thioesterase II 3.1.2.20 3.1.2.2 tesB Increase and ordecrease thioesterase III 3.1.2.— fadM Increase and or decreasehydroxyphenylacetyl-CoA N/A paaI Increase and or thioesterase decreaseesterase/thioesterase 3.1.2.28 ybgC Increase and or decreaseproofreading thioesterase in entH Increase and or enterobactinbiosynthesis decrease acetoacetyl-CoA synthase 2.3.1.194 npth07 Increase3-ketoacyl-CoA synthase/elongase 2.3.1 Elo1 Increase 3-ketoacyl-CoAsynthase/elongase 2.3.1 Elo2 Increase 3-Hydroxybutyryl-CoA dehydrogenase1.1.1.157 hbd Increase 3-oxoacyl-CoA reductase 1.1.1.100 fabG Increaseenoyl-CoA hydratase 4.2.1.17 crt Increase enoyl-CoA hydratase 4.2.1.17ech2 Increase Trans-2-enoyl-reductase 1.3.1.9 ter Increase thioesterase3.1.2.20 paaI Decrease E.C. No = “Enzyme Commission number”

Also, without being limiting, a first step in some multi-phase methodsof making a fatty acid may be exemplified by providing into a vessel,such as a culture or bioreactor vessel, a nutrient media, such as aminimal media as known to those skilled in the art, and an inoculum of agenetically modified microorganism so as to provide a population of suchmicroorganism, such as a bacterium, and more particularly a member ofthe family Enterobacteriaceae, such as E. coli, where the geneticallymodified microorganism comprises a metabolic pathway that convertsmalonyl-CoA to a fatty acid. This inoculum is cultured in the vessel sothat the cell density increases to a cell density suitable for reachinga production level of a fatty acid or fatty acid derived product thatmeets overall productivity metrics taking into consideration the nextstep of the method. In various alternative embodiments, a population ofthese genetically modified microorganisms may be cultured to a firstcell density in a first, preparatory vessel, and then transferred to thenoted vessel so as to provide the selected cell density. Numerousmulti-vessel culturing strategies are known to those skilled in the art.Any such examples provide the selected cell density according to thefirst noted step of the method.

Also without being limiting, a subsequent step may be exemplified by twoapproaches, which also may be practiced in combination in variousexamples. A first approach provides a genetic modification to thegenetically modified microorganism such that its enoyl-ACP reductaseenzymatic activity may be controlled. As one example, a geneticmodification may be made to substitute a temperature-sensitive mutantenoyl-ACP reductase (e.g., fabI^(TS) in E. coli) for the nativeenoyl-ACP reductase. The former may exhibit reduced enzymatic activityat temperatures above 30° C. but normal enzymatic activity at 30° C., sothat elevating the culture temperature to, for example to 34° C., 35°C., 36° C., 37° C. or even 42° C., reduces enzymatic activity ofenoyl-ACP reductase. In such case, more malonyl-CoA is converted to afatty acid than at 30° C., where conversion of malonyl-CoA to fattyacids is not impeded by a less effective enoyl-ACP reductase.

Other genetic modifications that may be useful in the production offatty acids may be included in the cell. For example, the ability toutilize sucrose may be provided, and this would expand the range of feedstocks that can be utilized to produce a fatty acid or fatty acidderived product or other chemical products. Common laboratory andindustrial strains of E. coli, such as the strains described herein, arenot capable of utilizing sucrose as the sole carbon source. Sincesucrose, and sucrose-containing feed stocks such as molasses, areabundant and often used as feed stocks for the production by microbialfermentation, adding appropriate genetic modifications to permit uptakeand use of sucrose may be practiced in strains having other features asprovided herein. Various sucrose uptake and metabolism systems are knownin the art (for example, U.S. Pat. No. 6,960,455).

Also, genetic modifications may be provided to add functionality forbreakdown of more complex carbon sources, such as cellulosic biomass orproducts thereof, for uptake, and/or for utilization of such carbonsources. For example, numerous cellulases and cellulase-based cellulosedegradation systems have been studied and characterized (Beguin, P andAubert, J-P, 1994; Ohima, K. et al., 1997.)

In some examples, genetic modifications increase the pool andavailability of the cofactor NADPH, and/or, consequently, theNADPH/NADP⁺ ratio may also be provided. For example, in E. coli, thismay be done by increasing activity, such as by genetic modification, ofone or more of the following genes: pgi (in a mutated form), pntAB,overexpressed, gapA:gapN substitution/replacement, and disrupting ormodifying a soluble transhydrogenase such as sthA, and/or geneticmodifications of one or more of zwf, gnd, and edd.

Any such genetic modifications may be provided to species not havingsuch functionality, or having a less than desired level of suchfunctionality. More generally, and depending on the particular metabolicpathways of a microorganism selected for genetic modification, anysubgroup of genetic modifications may be made to decrease cellularproduction of fermentation product(s) selected from the group consistingof acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters,isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene,isobutylene, ethyl acetate, vinyl acetate, other acetates,1,4-butanediol, 2,3-butanediol, butanol, isobutanol, sec-butanol,butyrate, isobutyrate, 2-OH-isobutryate, 3-OH-butyrate, ethanol,isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate,glucarate, glutarate, caprolactam, adipic acid, propanol, isopropanol,fusel alcohols, and 1,2-propanediol, 1,3-propanediol, formate, fumaricacid, propionic acid, succinic acid, valeric acid, and maleic acid. Genedeletions may be made as disclosed generally herein, and otherapproaches may also be used to achieve a desired decreased cellularproduction of selected fermentation products.

The first genetic mutation according to any aspect of the presentinvention may result in the formation of at least one fatty acid and/oracyl coenzyme A (CoA) thereof, wherein the fatty acid comprises at least5 carbon atoms. In particular, the fatty acid may be of any chain lengthfrom 5 to greater than 18 carbons. The fatty acid may be selected fromthe group consisting of: pentanoic acid, hexanoic acid, octanoic acid,decanoic acid, dodecanoic acid, myristic acid, palmitic acid,palmitoleic acid, stearic acid, and oleic acid. In particular, thesefatty acids may be produced from a fatty acyl-CoA intermediate via theactivity of a fatty acyl-CoA thioesterase. Alternatively, these fattyacids may be produced from a fatty acyl-CoA intermediate via concertedactivities of a fatty acyl-CoA phosphotransferase first producing afatty acyl-phosphate and then the action of a fatty acid kinaseoperating to produce a fatty acid from the fatty acyl-phosphate.

According to any aspect of the present invention the cell according toany aspect of the present invention may be combined with a carbon sourceto be able to produce the fatty acid. In particular, the carbon sourceprovided to the cell may have a ratio of carbon-14 to carbon-12 of about1.0×10⁻¹⁴ or greater. The carbon source may be selected from the groupconsisting of glucose, sucrose, fructose, dextrose, lactose, xylose,pentose, polyol, hexose, other cellulosic sugars or a combinationthereof. In one example, the carbon source may be glycerol. In anotherexample, the carbon source may be synthesis gas. Synthesis gas can forexample be produced as a by-product of coal gasification. Accordingly,the microorganism according to any aspect of the present invention maybe capable of converting a substance which is a waste product into avaluable resource.

In another example, synthesis gas may be a by-product of gasification ofwidely available, low-cost agricultural raw materials for use with themixed culture of the present invention to produce substituted andunsubstituted organic compounds. There are numerous examples of rawmaterials that can be converted into synthesis gas, as almost all formsof vegetation can be used for this purpose. In particular, raw materialsare selected from the group consisting of perennial grasses such asmiscanthus, corn residues, processing waste such as sawdust and thelike.

In general, synthesis gas may be obtained in a gasification apparatus ofdried biomass, mainly through pyrolysis, partial oxidation and steamreforming, wherein the primary products of the synthesis gas are CO, H₂and CO₂. Usually, a portion of the synthesis gas obtained from thegasification process is first processed in order to optimize productyields, and to avoid formation of tar. Cracking of the undesired tar andCO in the synthesis gas may be carried out using lime and/or dolomite.These processes are described in detail in for example, Reed, 1981.

In particular, the cell culture may comprise an inhibitor of fatty acidsynthase the cell may be genetically modified for reduced enzymaticactivity in the cell's fatty acid synthase pathway. This may allowbetter control for producing the specific desired fatty acid.

The cell according to any aspect of the present invention may compriseat least one second genetic mutation that may increase the activity ofat least one wax ester synthase in the cell relative to the wild typecell. The wax ester synthase may comprise sequence identity of at least50% to a polypeptide of SEQ ID NOs: 1-23 and combinations thereof or toa functional fragment of any of the polypeptides for catalyzing theconversion of fatty acid and/or acyl coenzyme A thereof to therespective fatty acid ester. In particular, the wax ester synthase maycomprise sequence identity of at least 50% to a polypeptide of SEQ IDNOs: 1-8 and combinations thereof. More in particular, the wax estersynthase used according to any aspect of the present invention maycomprise sequence identity of at least 60, 65, 70, 75, 80, 85, 90, 95,98 or 100% to a polypeptide of any one of sequences of SEQ ID NOs: 1-8and combinations thereof. These sequences are only reference amino acidsequences. In particular, the sequence of the wax ester synthase usedaccording to any aspect of the present invention may comprise aminoacids other than those essential for the function, for example thecatalytic activity of a protein, or the fold or structure of a moleculeare deleted, substituted or replaced by insertions or essential aminoacids are replaced in a conservative manner to the effect that thebiological activity of the reference sequence or a molecule derivedtherefrom is preserved. The state of the art comprises algorithms thatmay be used to align two given amino acid sequences and to calculate thedegree of identity, see Arthur Lesk (2008), and Katoh et al., 2005. Inparticular, the wax ester synthase sequences used according to anyaspect of the present invention may comprise the amino acids thatprovide the function to the protein. More in particular, the wax estersynthase sequences may comprise deletions, insertions or substitutionsin amino acid sequences as well as fusions that still retain thefunction of the wax ester synthase capable of catalyzing the conversionof fatty acid and/or acyl coenzyme A thereof to the respective fattyacid ester. In one example, a mutant of any one of the sequences of SEQID NO:1-8 may be used in any aspect of the present invention. A ‘mutant’used herein indicates a mutant derived from any one of the sequences ofSEQ ID NO:1-8 that is capable of maintaining the function of a wax estersynthase of converting a fatty acid and/or acyl coenzyme A thereof tothe respective fatty acid ester. Such a mutant has an amino acidsequence subjected to deletion, substitution, insertion, or addition ofat least one amino acid. The mutant of the present invention can beadequately produced with the use of any methods known to persons skilledin the art.

TABLE 2 Sequence of wax ester synthases Accession No. SEQ ID NO: (NCBI)Organism Sequence  1 522136843 Singularimonas MESPRTPMHVGGLMTFRLPPD(Svar) variicoloris APPDFLRQLFARLRAQMPSTEP FNLRLARTPWSALAPAWEPAPDIDIDYHVRHSALPYPGGEREL GVLVSRLHSHPLDLRRPPWEIT LIEGLENDRFAFFLKVHHSALDGMGALKLVRRWLSADALQRD MPALWALPAQPREARDAPHGH AVEQGVEALRTQLRASGELLSTLRRMARRRDNPEGGILSALSTP RTLLNVPITPQRRLATQLFELSR IKAVSAATQSTVNDVALALIAGAVRRYLLELDALPHEPLVASVP VGLPRADGKPGNAVAGFVVPL ETQADDPLDCLHVVRAVTQRTKDQLRGMSPEALAQFTMLGLS PLILGQMARVLSHLPPIFNFVVS NVVASKELLYLEGAELEAMYPISVLFDGYALNVTLVGYHDRLS LGFTGCRDALPSLQRLAVYSAE ALEELERAAGLVPHAAGAAEHAPARRTRRRGAH  2 YP_436128.1 Hahella MTPLSPVDQIFLWLEKRQQPM (Hche)chejuensis HVGGLHIFSFPDDADAKYMTEL KCTC 2396 AQQLRAYATPQAPFNRRLRQRWGRYYWDTDAQFDLEHHFRH EALPKPGRIRELLAHVSAEHSN LMDRERPMWECHLIEGIRGRRFAVYYKAHHCMLDGVAAMRM CVKSYSFDPTATEMPPIWAISK DVTPARETQAPAAGDLVHSLSQLVEGAGRQLATVPTLIRELGK NLLKARDDSDAGLIFRAPPSILN QRITGSRRFAAQSYALERFKAIGKAFQATVNDVVLAVCGSALR NYLLSRQALPDQPLIAMAPMSI RQDDSDSGNQIAMILANLGTHIADPVRRLELTQASARESKERFR QMTPEEAVNYTALTLAPSGLNL LTGLAPKWQAFNVVISNVPGPNKPLYWNGARLEGMYPVSIPVD YAALNITLVSYRDQLEFGFTAC RRTLPSMQRLLDYIEQGIAELEKAAGV  3 480024154 Acinetobacter MRPLHPIDFIFLSLEKRQQPMH (Ajun)junii NIPH 182 VGGLFLFEIPENASPTFVHDLVQ DIRQSKSIPVPPFNNQLNGLFWGEDPEFDIDHHFRHIALPNPGRI RELLVYISQQHSSLIDRAKPLW TCDIIEGIEGNRFAMYFKIHHAMVDGVAGMRLIEKSLSKDPNE KHVVPLWCVEGKRTKRLKAPK PPSVSKIKGIMDGIKSQLEVTPKVMQELSQTIFKEIGKNPDYVST FQAPPSILNQRVSSSRRFAAQSF ELDRFRNIAKSLGVTINDVVLAVCAGALREYLISHESLPKKPLIA MVPASLRTDDSDVSNRITMILA NLATHIEDPIERLQIIRRSVQNSKQRFSRMTANEILNYSALVYGPA GLNIVSGMLPKRQAFNLVISNV PGPREPLYWNGAKLDALYPASIVMDGQALNITMTSYLDKLEVG LIACRNALPKMQNLLTHLEDEI QRFESAILSLPKQAAEG  4449424446 Amycolatopsis MPFMPVTDSMFLLVETREHPM (Aazu) azurea DSMHVGGLQLFKKPEDAGPDYLRD 43854 LRRKLLDSDNMRDVFRRRPAR PVNTAGHVAWATDNDLELDYHFRHSALPQPGRIRELLELTGR WHSTLLDRHRPLWEIHLVEGL QDGRFAIYSKIHHALMDGVSALRHLQGTLSDDPTDLDCPPPWGR RPKPDGGRNGKASPSVLSTFGK TVNQLAGIAPAAMKVAREAFQEHTLTLPAQAPKTMLNVPIGGA RRFAAQSWSLDRVRKVATAAG VSRNDVVLAMCSGALRDYLIEQNSLPDAPLTAMVPVSLRRKDS GDAAGNNIGALLCNLATHLTD PAARLATINASMRNGKKLFSELTPLQTLLLSGINVAQLGVSPIPG FVNNTKPPFNLVISNVPGPRKQ MYWNGASLDGIYPASVLLDGQALNITLTSNGDNLDFGVTGCRR SVPHLQRILTHLDTALAELEHA VSVGRS  5 479966651Acinetobacter MRPLHPIDFIFLSLEKRQQPMH (Acip) sp. CIP 56.2VGGLFLFELPENASPTFVHDLV NEIRQSKSIPVPPFNNQLNGLFW GEDSEFDLDHHFRHIALPNPGRIRELLVYISQQHSSLIDRAKPLW TCDIIEGIEGNRFAMYFKIHHA MVDGVAGMRLIEKSLSQDPNEKHVVPLWCVEGKRTKRLKAPK PPTVSKIKGVMEGIKSQLEVAP KVMQELSQTIFKEMGKNPDYVSTFQAPPSILNQRVSSSRRFAAQ SFELGRFRRIAKSLGVTLNDVIL AVCSGALREYLISHNSLPKKPLIAMVPASLRTDDSDVSNRITMIL ANLATHIEDPIERLEVIRRSVQN SKQRFSRMTANEILNYSAVVYGPAGLNIASGMLPKRQAFNLVIS NVPGPREPLYWNGAKLDALYP ASIVMDGQALNITMTSYLDKLEVGLIACRNALPKMQNLLTHLEE EIQRFEQAIQDLPQKVAN  6 ABO21021 MarinobacterMKRLGTLDASWLAVESEDTPM hydrocarbono- HVGTLQIFSLPEGAPETFLRDMclasticus ATCC VTRMKEAGDVAPPWGYKLAW 49840 SGFLGRVIAPAWKVDKDIDLDYHVRHSALPRPGGERELGILVSR LHSNPLDFSRPLWECHVIEGLE NNRFALYTKMHHSMIDGISGVRLMQRVLTTDPERCNMPPPWT VRPHQRRGAKTDKEASVPAAV SQAMDALKLQADMAPRLWQAGNRLVHSVRHPEDGLTAPFTGP VSVLNHRVTAQRRFATQHYQL DRLKNLAHASGGSLNDIVLYLCGTALRRFLAEQNNLPDTPLTAG IPVNIRPADDEGTGTQISFMIAS LATDEADPLNRLQQIKTSTRRAKEHLQKLPKSALTQYTMLLMS PYILQLMSGLGGRMRPVFNVTI SNVPGPEGTLYYEGARLEAMYPVSLIAHGGALNITCLSYAGSLN FGFTGCRDTLPSMQKLAVYTG EALDELESLILPPKKRARTRK  7YP_957462 Marinobacter MGSSHHHHHHSSGLVPRGSHM (Maqu aquaeolei VT8TPLNPTDQLFLWLEKRQQPMH T373M, T373M, Q420R VGLQLFSFPEGAPDDYVAQLA Q420R)DQLRQKTEVTAPFNQRLSYRLG QPVWVEDEHLDLEHHFRFEAL PTPGRIRELLSFVSAEHSHLMDRERPMWEVHLIEGLKDRQFALY TKVHHSLVDGVSAMRMATRM LSENPDEHGMPPIWDLPCLSRDRGESDGHSLWRSVTHLLGLSG RQLGTIPTVAKELLKTINQARK DPAYDSIFHAPRCMLNQKITGSRRFAAQSWCLKRIRAVCEAYG TTVNDVVTAMCAAALRTYLM NQDALPEKPLVAFVPVSLRRDDSSGGNQVGVILASLHTDVQEAG ERLLKIHHGMEEAKQRYRHMS PEEIVNYTALTLAPAAFHLLTGLAPKWQMFNVVISNVPGPSRPL YWNGAKLEGMYP VSIDMDRLALNMTLTSYNDRVEFGLIGCRRTLPSLQRMLDYLE QGLAELELNAGL  8 YP_957462 MarinobacterMGSSHHHHHSSGLVPRGSHMT (Maqu aquaeolei VT8 PLNPTDQLFLWLEKRQQPMHV E72K)E72K (no final GGLQLFSFPEGAPDDYVAQLA H in His-tag)DQLRQKTEVTAPFNQRLSYRLG QPVWVKDEHLDLEHHFRFEAL PTPGRIRELLSFVSAEHSHLMDRERPMWEVHLIEGLKDRQFALY TKVHHSLVDGVSAMRMATRM LSENPDEHGMPPIWDLPCLSRDRGESDGHSLWRSVTHLLGLSG RQLGTIPTVAKELLKTINQARK DPAYDSIFHAPRCMLNQKITGSRRFAAQSWCLKRIRAVCEAYG TTVNDVVTAMCAAALRTYLM NQDALPEKPLVAFVPVSLRRDDSSGGNQVGVILASLHTDVQEAG ERLLKIHHGMEEAKQRYRHMS PEEIVNYTALTLAPAAFHLLTGLAPKWQTFNVVISNVPGPSRPL YWNGAKLEGMYPVSIDMDRL ALNMTLTSYNDQVEFGLIGCRRTLPSLQRMLDYLEQGLAELELN AGL 23 (Maqu) YP_957462 MarinobacterMTPLNPTDQLFLWLEKRQQPM aquaeolei VT8 HVGGLQLFSFPEGAPDDYVAQLADQLRQKTEVTAPFNQRLSYR LGQPVWVEDEHLDLEHHFRI-th ALPTPGRIRELLSFVSAEHSHLMDRERPMWEVHLIEGLKDRQFA LYTKVHHSLVDGVSAMRMAT RMLSENPDEHGMPPIWDLPCLSRDRGESDGHSLWRSVTHLLGL SGRQLGTIPTVAKELLKTINQA RKDPAYDSIFHAPRCMLNQKITGSRRFAAQSWCLKRIRAVCEA YGTTVNDVVTAMCAAALRTYL MNQDALPEKPLVAFVPVSLRRDDSSGGNQVGVILASLHTDVQE AGERLLKIHHGMEEAKQRYRH MSPEEIVNYTALTLAPAAFHLLTGLAPKWQTFNVVISNVPGPSR PLYWNGAKLEGMYPVSIDMDR LALNMTLTSYNDQVEFGLIGCRRTLPSLQRMLDYLEQGLAELEL NAGL

Throughout this application, any data base code, unless specified to thecontrary, refers to a sequence available from the NCBI data bases, morespecifically the version online on 12 Jun. 2014, and comprises, if suchsequence is a nucleotide sequence, the polypeptide sequence obtained bytranslating the former.

The cell according to any aspect of the present invention may comprise athird genetic mutation that reduces the fatty acid degradation capacityof the cell relative to the wild type cell.

Degradation of fatty acids is accomplished by a sequence ofenzymatically catalyzed reactions. First of all, fatty acids are takenup and translocated across the cell membrane via atransport/acyl-activation mechanism involving at least one outermembrane protein and one inner membrane-associated protein which hasfatty acid-CoA ligase activity, referred to in the case of E. coli asFadL and FadD/FadK, respectively. Inside the cell, the fatty acid to bedegraded is subjected to enzymes catalyzing other reactions of theβ-oxidation pathway. The first intracellular step involves theconversion of acyl-CoA to enoyl-CoA through acyl-CoA dehydrogenase, thelatter referred to as FadE in the case of E. coli. The activity of anacyl-CoA dehydrogenase may be assayed as described in the state of art,for example by monitoring the concentration of NADHspectrophotometrically at 340 nm in 100 mM MOPS, pH 7.4, 0.2 mMEnoyl-CoA, 0.4 mM NAD⁺. The resulting enoyl-CoA is converted to3-ketoacyl-CoA via 3-hydroxylacyl-CoA through hydration and oxidation,catalyzed by enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase,referred to as FadB and FadJ in E. coli. Enoyl-CoAhydratase/3-hydroxyacyl-CoA dehydrogenase activity, more specificallyformation of the product NADH may be assayed spectrophotometrically asdescribed in the state of the art, for example as outlined for FadE.Finally, 3-ketoacyl-CoA thiolase, FadA and FadI in E. coli, catalyzesthe cleavage of 3-ketoacyl-CoA, to give acetyl-CoA and the inputacyl-CoA shortened by two carbon atoms. The activity of ketoacyl-CoAthiolase may be assayed as described in the state of the art, forexample in Antonenkov, V., D. et al. (1997) Substrate specificities of3-oxoacyl-CoA thiolase and sterol carrier protein 2/3-oxoacyl-coAthiolase purified from normal rat liver peroxisomes. Sterol carrierprotein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of2-methyl-branched fatty acids and bile acid intermediates. In oneexample, the term “a cell having a reduced fatty acid degradationcapacity”, as used herein, refers to a cell having a reduced capabilityof taking up and/or degrading fatty acids. The fatty acid degradationcapacity of a cell may be reduced in various ways. In another example,the cell has, compared to its wild type, a reduced activity of an enzymeinvolved in the β-oxidation pathway. In a further example, the term“enzyme involved in the β-oxidation pathway”, as used herein, refers toan enzyme that interacts directly with a fatty acid or a derivativethereof formed as part of the degradation of said fatty acid via theβ-oxidation pathway the sequence of reactions effecting the conversionof a fatty acid to acetyl-CoA and the CoA ester of the shortened fattyacid, for example by recognizing the fatty acid or derivative thereof asa substrate, and converts it to a metabolite formed as a part of theβ-oxidation pathway. More in particular, the reduced fatty aciddegradation capacity in the cell according to any aspect of the presentinvention may be the third genetic mutation which results in a decreasein the expression of at least one enzyme selected from the groupconsisting of acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase,enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase relative to the wildtype cell.

For example, the acyl-CoA dehydrogenase is an enzyme involved in theβ-oxidation pathway as it interacts with fatty acid-CoA and convertsfatty acid-CoA ester to enoyl-CoA, which is a metabolite formed as partof the β-oxidation. In a further example, the term “enzyme involved inthe β-oxidation pathway”, as used herein, comprises any polypeptide fromthe group comprising acyl-CoA dehydrogenase, enoyl-CoA hydratase,3-hydroxyacyl-CoA dehydrogenase and 3-keto-acyl-CoA thiolase.Subsequently, the acyl-CoA synthetase may catalyze the conversion afatty acid to the CoA ester of a fatty acid, i.e. a molecule, whereinthe functional group —OH of the carboxy group is replaced with —S-CoA,for introducing said fatty acid into the β-oxidation pathway. Forexample, the polypeptides FadD and FadK in E. coli (access code:BAA15609.1) are acyl-CoA dehydrogenases. In an example, the term“acyl-CoA dehydrogenase”, as used herein, is a polypeptide capable ofcatalyzing the conversion of an acyl-CoA to enoyl-CoA, as part of theβ-oxidation pathway. For example, the polypeptide FadE in E. coli(access code: BAA77891.2) is an acyl-CoA dehydrogenase. In one example,the term “2,4-dienoyl-CoA reductase”, as used herein, is a polypeptidecapable of catalyzing the conversion of the 2,4-dienoyl CoA from anunsaturated fatty acid into enoyl-CoA, as part of the β-oxidationpathway. For example, the polypeptide FadH in E. coli is a2,4-dienoyl-CoA reductase. In an example, the term “enoyl-CoAhydratase”, as used herein, also referred to as 3-hydroxyacyl-CoAdehydrogenase, refers to a polypeptide capable of catalyzing theconversion of enoyl-CoA to 3-ketoacyl-CoA through hydration andoxidation, as part of the β-oxidation pathway. For example, thepolypeptides FadB and FadJ in E. coli (access code: BAE77457.1) areenoyl-CoA hydratases. In one example, the term “ketoacyl-CoA thiolase”,as used herein, refers to a polypeptide capable of catalyzing theconversion of cleaving 3-ketoacyl-CoA, resulting in an acyl-CoAshortened by two carbon atoms and acetyl-CoA, for example as the finalstep of the β-oxidation pathway. For example, the polypeptides FadA andFadI in E. coli (access code: AP009048.1) are ketoacyl-CoA thiolases.

In particular, the cell according to any aspect of the present inventionmay comprise genetic mutations that result in an increase in theexpression of β-ketoacyl-ACP synthase III. This may be the firstmutation in the cell according to any aspect of the present invention.Any β-ketoacyl-ACP synthase III (fabH) known in the art may be used inthe method according to any aspect of the present invention. Inparticular, the fabH may be selected from Table 3a.

TABLE 3a Possible sources of FabH that may be used to produce fattyacids such as lauric acid. FabH Accession No. Shewanella sp. MR-4gi|113969844 Shewanella frigidimarina NCIMB 400 gi|114563637 Shewanellasp. ANA-3 gi|117920011 Shewanella amazonensis SB2B gi|119774647Shewanella sp. W3-18-1 gi|120598458 Shewanella baltica OS185gi|153001200 Gordonia bronchialis DSM 43247 gi|262201496 Gordonianeofelifaecis NRRL B- gi|326383808 59395 putative Shewanella sp. HN-41gi|336312046 Rheinheimera sp. A13L gi|336314652 Gordonia araii NBRC100433 gi|359421305 Gordonia polyisoprenivorans NBRC gi|359767552 16320Gordonia effusa NBRC 100432 gi|359774344 Alishewanella jeotgali KCTC22429 gi|375108677 Gordonia otitidis NBRC 100426 gi|377561073 Gordoniasputi NBRC 100414 gi|377565709 Gordonia terrae NBRC 100016 gi|377571475Gordonia polyisoprenivorans VH2 gi|378716896 Alishewanella agri BL06gi|393761603 Gordonia sp. KTR9 gi|404214055 Shewanella oneidensis MR-1gi|414562081 Gordonia hirsuta DSM 44140 gi|441517717 Gordonia sihwensisNBRC 108236 gi|441522685 Gordonia soli NBRC 108243 gi|444431726 Gordoniamalaquae gi|495656093 Gordonia sp. NB4-1Y gi|464805365 Thalassolituusoleivorans MIL-1 gi|473830078 Colwellia psychrerythraea 34H gi|71278947Shewanella denitrificans OS217 gi|91793871 Rheinheimera nanhaiensisE407-8 gi383934006 Acinetobacter sp. CIP 53.82 480152603 Hahellachejuensis KCTC 2396 83645428 Acinetobacter sp. SH024 293608659Acinetobacter sp. NBRC 10098 359430113 Acinetobacter sp. ADP1 50085221Acinetobacter ursingii DSM 16037 = 406040759 CIP 107286 Acinetobacterbohemicus ANC 3994 479867614 Candidatus Accumulibacter phosphatis257092603 clade IIA str. UW-1 blood disease bacterium R229 344167953Ralstonia solanacearum CMR15 410684104 Marinobacter sp. BSs20148399545195 Marinobacter algicola DG893 149375225 Ralstonia sp. 5_7_47FAA309779507 Rubrivivax gelatinosus IL144 383757692 Oceanobacter sp. RED6594501061 gamma proteobacterium HTCC5015 254447852 Ilumatobactercoccineus YM16-304 470180366 marine gamma proteobacterium 254480565HTCC2148 Marinobacter aquaeolei VT8 120554511 Alcanivorax sp. DG881254427265 Hydrocarboniphaga effusa AP103 392950783 Curvibacter putativesymbiont of Hydra 260220470 magnipapillata gamma proteobacterium HdN1304312991 marine gamma proteobacterium 119503170 HTCC2080 gammaproteobacterium IMCC3088 329897271 gamma proteobacterium NOR5-3254514195 Acinetobacter radioresistens SK82 255318218 Acinetobacter sp.NIPH 899 479953276 Acinetobacter schindleri CIP 107287 480002578Acinetobacter towneri DSM 14962 = 480029713 CIP 107472 Acinetobacterjunii CIP 107470 480007780 Acinetobacter sp. CIP 56.2 479964140Acinetobacter baumannii AYE 169796586 baumannii MDR-ZJ06 Acinetobactergerneri DSM 14967 = 479991204 CIP 107464 Acinetobacter bouvetii DSM14964 = 480043238 CIP 107468 Acinetobacter sp. ANC 3789 479932652Acinetobacter lwoffii SH145 262375396 Acinetobacter soli NIPH 2899480019083 Acinetobacter baumannii WC-323 425744072 Acinetobactercalcoaceticus NIPH 13 479856262 Acinetobacter johnsonii SH046 262369694Acinetobacter haemolyticus CIP 64.3 480080132 Acinetobacter sp. CIP102529 479942708 Acinetobacter sp. CIP-A165 479879420 Acinetobacterguillouiae CIP 63.46 479909393 Ralstonia solanacearum FQY_4 469776065Ralstonia solanacearum UW551 83747353

The β-ketoacyl-ACP synthase III (FabH) may comprise sequence identity ofat least 50% to a polypeptide selected from the group consisting of SEQID NOs: 24-27 and combinations thereof or to a functional fragment ofany of the polypeptides. In particular, the FabH may comprise sequenceidentity of at least 50% to a polypeptide of SEQ ID NOs: 24-27 andcombinations thereof. More in particular, the FabH used according to anyaspect of the present invention may comprise sequence identity of atleast 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% to a polypeptide of anyone of sequences of SEQ ID NOs: 24-27 and combinations thereof. More inparticular, the cell according to any aspect of the present inventionmay have a first mutation that comprises a combination of sequences ofFabH. For example, the cell according to any aspect of the presentinvention may be genetically modified to comprise a polypeptide withsequence identity of at least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98 or100% to a polypeptide of comprising SEQ ID NOs: 24 and 27, SEQ ID NOs:25 and 27, SEQ ID NOs: 26 and 27 and the like.

These sequences are only reference amino acid sequences. In particular,the sequence of the FabH used according to any aspect of the presentinvention may comprise amino acids other than those essential for thefunction, for example the catalytic activity of a protein, or the foldor structure of a molecule are deleted, substituted or replaced byinsertions or essential amino acids are replaced in a conservativemanner to the effect that the biological activity of the referencesequence or a molecule derived therefrom is preserved.

TABLE 3bSequences of FabH, crt, Hbd that may be used in the cells according to any aspect of the present invention SEQ ID Accession No. NO: (NCBI)/Organism Sequence 24 WP_014577218.1| Marinobacter MIKAVISGTGLYTPPATISNDEadhaerens LVEAFNQYVELFNAENADAI HP15 ASGDVTPLQPSSSSFIEKASGIKRRHVIDKDGILDPNRMKPYI PDRSNEEPSVQCDMAVTACR EALEQAGKSAEDVDAVIVACSNLQRAYPAVSIEVQEALGID GFAYDMNVACSSATFGLQAA VNSVENGSARAVLVVSPEICSGHLNFRDRDSHFIFGDACTAI LVEREEDTREGQGFEILGTSL KTKFSNNIRNNFGFLNRADESGVGKPDKLFVQQGRKVFKEV SPLVAETIQKQLQSLSLAPDD LRRMWLHQANLNMNQLIARKVLGRDATEEEAPVILDEYAN TSSAGSIIAFHKNKDDLVSGD LGVICSFGAGYSIGSVVVRRR 25ZP_10350240 Alishewanella MQQVVISGSGLFTPQHRISNE agri BL06ELVQSYNQYVDQFNEEHAAA IAAGEIEALEYSSTEFIEKASGI KARHVLYKDGILDPKIMHPVFRKRGEDELPEMVEMAVQAA TQALAQANKTAADIDLIICAA SNMQRPYPALSVELQQALGAGGYAFDMNVACSSATFAISN AVNAIRGGTAKVVLVVNPEF ASPQVDYRSRDSHFIFGDVCTATIIEAESSCSSQQAFRILGMR LKTTFSNNIRCDIGYTEHCFTE QDPKAPFFKQQGRKVFKELLPIVADVIQDEMAAQNLAPDDL KRLWLHQANINMNIFAAKKI LGRDPLPEEAPLVLDTYANTASAGSIIAFHKYQQGLVSGDKA ILCSFGAGYSVGCVVLEKC 26 ENU26638 AcinetobacterMGIRITGTGLFHPTESISNEEL sp. NIPH 236 VESLNAYVEQFNQENAEQIAAGEIEALRGSSPEFIEKASGIQ RRYVVEKSGILDPKRLRPRLQ ERSNDELSLQAEWGVIAAKQAMENAGVTAEDIDVVILACS NMQRAYPAVAIEIQSALGIQG YAYDMNVACSAATFGLKQAYDAVKCGARRVLLLNVEITS GHLDYRTRDAHFIFGDVATA SIIEETETKSGYEILDIHLFTQFSNNIRNNFGFLNRSEDAVVDD KLFRQDGRKVFKEVCPLVAKI ITAQLEKLELTPEQVKRFWLHQANANMNELILKLVVGKEAD LERAPIILDEFANTSSAGVIIA MHRTGEQVNNGEYAVISSFGAGYSVGSIIVQKHIA 27 YP_006031367 Ralstonia MHDVVISGTGLWVAPEVITNsolanacearum EELVASFNAYARHYNEANAT Po82 AIAAGTLAAVAESSVEFIEKASGIRQRYVIDKAGVLDPARM RPRLAPRGDDALSLQAEIGVA AAREALAAAGRDAGDIDMLICSAANMQRPYPAMGIEIQNA LGADGYAFDMNVACSSATFG LEQAINAVRTGSARVALMVNPEITSGHLAWKDRDCHFIFGD VCTAVVVERADDARAPDQW QVLGTRMATRFSNSIRNNAGFLSRSEDRDPDDRDQLFRQEGR KVFKEVCPMAAEHIAGHLQS LGHAPADVRRFWLHQANLGMNQLIGKRLLGRDASADEAP VILDEFANTASAGSIIAFHRHR ADLQPGDLGLICSFGAGYSIGSVAVRKR 28 AAA95967 Clostridium MELNNVILEKEGKVAVVTIN acetobutylicumRPKALNALNSDTLKEMDYVI ATCC 824 GEIENDSEVLAVILTGAGEKSFVAGADISEMKEMNTIEGRKF GILGNKVFRRLELLEKPVIAA VNGFALGGGCEIAMSCDIRIASSNARFGQPEVGLGITPGFGG TQRLSRLVGMGMAKQLIFTA QNIKADEALRIGLVNKVVEPSELMNTAKEIANKIVSNAPVAV KLSKQAINRGMQCDIDTALAF ESEAFGECFSTEDQKDAMTAFIEKRKIEGFKNR 29 NP_349314 Clostridium MKKVCVIGAGTMGSGIAQAFacetobutylicum AAKGFENVLRDIKDEFVDRG ATCC 824 LDFINKNLSKLVKKGKIEEATKVEILTRISGTVDLNMAADCD LVIEAAVERMDIKKQIFADLD NICKPETILASNTSSLSITEVASATKRPDKVIGMHFFNPAPVM KLVEVIRGIATSQETFDAVKE TSIAIGKDPVEVAEAPGFVVNRILIPMINEAVGILAEGIASVE DIDKAMKLGANHPMGPLELG DFIGLDICLAIMDVLYSETGDSKYRPHTLLKKYVRAGWLGR KSGKGFYDYSK

As used herein, the term “fatty ester” means an ester. In particular, afatty ester is any ester made from a fatty acid to produce a fatty acidester. In one example, a fatty ester contains an A side (i.e., thecarbon chain attached to the carboxylate oxygen) and a B side (i.e., thecarbon chain comprising the parent carboxylate). In a particular, whenthe fatty ester is derived from the fatty acid biosynthetic pathway, theA side is contributed by an alcohol, and the B side is contributed by afatty acid. Any alcohol can be used to form the A side of the fattyesters. For example, the alcohol can be derived from the fatty acidbiosynthetic pathway. Alternatively, the alcohol can be produced throughnon-fatty acid biosynthetic pathways. In one example, the alcohol can beprovided exogenously. For example, the alcohol can be supplied in thefermentation broth in instances where the fatty ester is produced by anorganism that can also produce the fatty acid. Alternatively, acarboxylic acid, such as a fatty acid or acetic acid, can be suppliedexogenously in instances where the fatty ester is produced by anorganism that can also produce alcohol.

The carbon chains comprising the A side or B side can be of any length.In one example, the A side of the ester is at least about 1, 2, 3, 4, 5,6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. The B side of theester is at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26carbons in length. The A side and/or the B side can be straight orbranched chain. The branched chains may have one or more points ofbranching. The branched chains may also include cyclic branches and/orthe A side and/or B side can be saturated or unsaturated. Ifunsaturated, the A side and/or B side can have one or more points ofunsaturation.

In one example, the fatty ester according to any aspect of the presentinvention may be produced biosynthetically. In particular, the fattyacid may be “activated” to produce at least one compound selected fromthe group consisting of acyl Coenzyme A (acyl-CoA), and acyl phosphate.More in particular, the fatty ester may be activated to Acyl-CoA, adirect product of fatty acid biosynthesis or degradation. Acyl-CoA mayalso be synthesized from a free fatty acid, a CoA, or an adenosinenucleotide triphosphate (ATP). An example of an enzyme which producesacyl-CoA is acyl-CoA synthase. Acyl-CoA may then be transferred to arecipient nucleophile such as alcohols, thiols, phosphates and the like.

The cell according to any aspect of the present invention may be furthergenetically modified to increase the expression of 3-hydroxyacylcoenzyme A dehydratase (3HCDh) and/or keto acyl-CoA reductase (KCR)relative to the wild type cell. This is increase in expression mayimprove the activity of fadB. In particular, the 3HCDh maycrotonase/enoyl-CoA hydratase (Crt) and/or the KCR may be hydroxybutyricdehydrogenase (Hbd). More in particular, the Crt may have sequenceidentity of at least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100%identity to a polypeptide of SEQ ID NO:28 and/or the Hbd has sequenceidentity of at least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100%identity to a polypeptide of SEQ ID NO:29.

In particular, the fatty acid ester may be produced in the presence ofat least one exogenous alcohol selected from the group consisting ofmethanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol,octanol, decanol, dodecanol, tetradecanol, hexadecanol and the like.

More in particular, the fatty acid may be 12 carbons in length. Thefatty acid may be lauric acid, the acyl coenzyme A thereof may belauroyl coenzyme A and the fatty acid ester may be methyl laurate.According to another aspect of the present invention there is provided amethod for producing methyl laurate, the method comprising contactinglauric acid and/or lauroyl coenzyme A with an isolated wax estersynthase that has sequence identity of at least 50% to a polypeptide ofSEQ ID NOs: 1-8 and combinations thereof. More in particular, the waxester synthase used according to any aspect of the present invention maycomprise sequence identity of at least 60, 65, 70, 75, 80, 85, 90, 95,98 or 100% to a polypeptide of any one of sequences of SEQ ID NOs: 1-8and combinations thereof. These sequences are only reference amino acidsequences. In particular, the sequence of the wax ester synthase usedaccording to any aspect of the present invention may comprise aminoacids other than those essential for the function, for example thecatalytic activity of a protein, or the fold or structure of a moleculeare deleted, substituted or replaced by insertions or essential aminoacids are replaced in a conservative manner to the effect that thebiological activity of the reference sequence or a molecule derivedtherefrom is preserved.

In particular, the method according to any aspect of the presentinvention is carried out within the cell according to any aspect of thepresent invention.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting unless otherwise specified.

The foregoing describes preferred embodiments, which, as will beunderstood by those skilled in the art, may be subject to variations ormodifications in design, construction or operation without departingfrom the scope of the claims. These variations, for instance, areintended to be covered by the scope of the claims.

EXAMPLES Example 1

Optimization of C12 Fatty Acyl-CoA Production in E. coli

Production of Fatty Acids Via Malonyl-CoA and Acetyl-CoA in a ShakeFlask Experiment

TABLE 4a List of microorganism strains that were used to produce fattyacids in the subsequent examples. The method of production and thesequences of the strains are provided in Table 3.2 of WO2014026162A1(OPX Biotechnologies Inc., USA). Strain designation Host Plasmid SEQ IDNOs. BXF_0012 BX_864 1)pBMT-3_ccdAB 30 BXF_0013 BX_8641)pBMT-3_ccdAB_P_(T7)- 31 ′tesA BXF_0014 BX_864 1)pBMT-3_ccdAB_P_(T7)-32 nphT7-hbd-crt-ter BXF_0015 BX_864 1)pBMT-3_ccdAB_P_(T7)- 33′tesA_PT7-nph_(T7)-hbd-crt- ter BXF_0018 BX_864 pBMT-3_ccdAB_PT7- 32nphT7-hbd-crt-ter BXF_0020 BX_860 1)pBMT-3_ccdAB_PT7- 33′tesA_PT7-nphT7-hbd-crt- ter BXF_0021 BX_876 1)pBMT-3_ccdAB_PT7- 33′tesA_PT7-nphT7-hbd-crt- ter BXF_0022 BX_874 1)pBMT-3_ccdAB 30 BXF_0023BX_874 1)pBMT-3_ccdAB_PT7- 31 ′tesA BXF_0024 BX_874 1)pBMT-3_ccdAB_PT7-33 ′tesA_PT7-nphT7-hbd-crt- ter BXF_0025 BX_875 1)pBMT-3_ccdAB 30BXF_0026 BX_875 1)pBMT-3_ccdAB_PT7- 31 ′tesA BXF_0027 BX_8751)pBMT-3_ccdAB_PT7- 33 ′tesA_PT7-nphT7-hbd-crt- ter BXF_0028 BX_8781)pBMT-3ccdAB-T7- 33 ′tesA- PT7_nphT7_hbd_crt_ter BXF_0028 BX_8781)pBMT-3_ccdAB_PT7- 33 ′tesA_PT7-nphT7-hbd-crt- ter BXF_0029 BX_8791)pBMT-3_ccdAB_PT7- 33 ′tesA_PT7-nphT7-hbd-crt- ter BXF_0030 BX_8811)pBMT-3_ccdAB_PT7- 33 ′tesA_PT7-nphT7-hbd-crt- ter BXF_0031 BX_8641)pBMT-3_ccdAB_PT7- 33 ′tesA_PT7-nphT7-hbd-crt- ter 2)pET-28b(emptyvector) BXF_0033 BX_878 1)pBMT-3_ccdAB_PT7- 32 nphT7-hbd-crt-terBXF_0034 BX_879 2)pBMT-3_ccdAB_PT7- 32 nphT7-hbd-crt-ter

A base strain was constructed by chromosomal integration of Hbd and Crtinto the BX_1018 parent strain. The following set of strains weretransformed and evaluated in small scale for both FAME production andmetabolite accumulation.

TABLE 4b list of strains used for testing hbd and crt presence in FAMEproduction Strain Parent(s) Plasmid 1 Plasmid 2 BXE_062 BX_1018pET-PpstsIH-Aagr pACYC-PpstsIH-nphT7(SV)-ter- PpstsIH-fadB-Maqu BXE_207BX_1018 pET-PpstsIH-Aagr pACYC-PpstsIH-nphT7(SV)-ter-PpstsIH-fadB-PphoE-Maqu BXE_229 BX_1018 pET-PpstsIH-AagrpACYC-PpstsIH-nphT7(SV)-ter- ΔyibD:PyibD-hbd-crt PpstsIH-fadB-PphoE-MaquBXE_230 BX_1018 pET-PpstsIH-Aagr pACYC-PpstsIH-nphT7(SV)-ter-ΔyibD:PyibD-hbd-crt PpstsIH-fadB-Maqu

To better understand the impact of Hbd/Crt expression, metaboliteaccumulation was monitored in cell lysate following incubation withmalonyl-CoA and acetyl-CoA substrates. The results of this assay, aspresented in FIG. 12, showed increased accumulation of C4-C6-CoAintermediates consistent with the expected activity on keto- andhydroxy-C4-CoAs.

Engineering β-Keto Acyl-CoA Synthases (FabH)

A screening approach was employed for β-keto acyl-CoA synthase homologsto identify candidates for lauric acid production based on demonstratedactivity on C4- to C10 acyl-CoA substrates. Greater than 70 homologshave been synthesized, expressed, purified and evaluated for activity invitro.

Synthase candidates identified to have significant activity on C4- toC10-CoA substrates were incorporated into production hosts and evaluatedfor FFA production in shake flask. FIG. 1 shows FFA production profilesat 68 hours for several of the engineered strains (Table 4c) exhibitingsignificant lauric acid production. As shown, both lauric acid and totalFFA production profiles are modulated by the individual synthasecandidate(s) incorporated. For example, strain BXF_198, which containsthe Aagr fabH construct, shows the highest specificity for lauric acid,whereas co-expression of Rsol fabH corresponds to the highest titer intotal FFA. All four synthase combinations shown in FIG. 1 have beenselected for Example 3 focused on production of methyl laurate.

TABLE 4c Bacterial strains with specific plasmids used in the screeningphase Strain Host Plasmid 1 Plasmid 2 BXF_166 BX_978 1009_pACYC-PpstsIH-1007_pET28b- nphT7(I147S,F217V)-ter- ΔlacI-PpstsIH-Madh TT_PpstsIH-fadBfabH-Aagr fab H BXF_169 BX_985 1009_pACYC-PpstsIH- 1008_pET28b-nphT7(I147S,F217V)-ter- ΔlacI-PpstsIH-Anip TT_PpstsIH-fadB fabH-Aagr fabH BXF_185 BX_985 1009_pACYC-PpstsIH- 1045_pET28b-nphT7(I147S,F217V)-ter- ΔlacI-PpstsIH-Rsol TT_PpstsIH-fadB fabH-Aagr fabH BXF_198 BX_985 1009_pACYC-PpstsIH- 1123_pET28b-nphT7(I147S,F217V)-ter- ΔlacI-PpstsIH-Aagr TT_PpstsIH-fadB fab H

Baseline FFA Analysis

A more comprehensive baseline analysis was completed with a subset ofFFA production strains with various synthase combinations as describedabove. Data for strain BXF_169 is presented; similar trends wereobserved among all strains that were evaluated in the more comprehensivetests. The data presented in FIG. 2 shows a time course of lauric acidproduction for strain BXF_169. In the shake flask test conditions, thestrain exhibited a stable initial production rate for 10-20 hours. Afterthe 20 hours, the rate decreased significantly and product titer levelsoff.

For all strains analyzed, samples were taken at each time point andanalyzed for transcript, expression, and activity for key enzymes in thefatty acid production pathway. FIGS. 3 and 4 show the relative mRNAexpression of the pathway genes and the corresponding enzyme activityover time for BXF_169. Transcription levels were good, but also differedbetween the five genes despite being driven from the same promoter. Bothtranscript and enzyme activity showed a decreasing trend over time, withenzyme activities dropping for several reactions at the 24 hour timepoint. Similar trends were observed for all other FFA production strainsevaluated.

In addition to reduced mRNA and enzyme activity, the 24-hour time pointwas also characterized by increased insoluble protein accumulation anddecreased glucose consumption for all strains (data not shown). As fattyacids including lauric acid are known to accumulate inside the cells inthe absence of specific transporters, it was hypothesized that thereduction in productivity at 24 hours is due to intracellular FFAtoxicity.

In Vitro Analysis of the Fatty Acid Synthesis Pathway

An assay was developed by reconstructing the fatty acyl-CoA pathway invitro with purified enzymes. The in vitro reconstruction simplifiedidentification of rate-limiting steps, which were characterized bymetabolite accumulation under reaction conditions. There were severalbenefits of in vitro pathway reconstitution including isolation ofpathway flux from competitive enzymes (e.g. thioesterases), substratelimitations (e.g. NADH pools), and balanced expression of multiplepathway enzymes. The equilibrium of the pathway was evaluated byquantifying all 20 pathway intermediates from malonyl-CoA to lauroyl-CoAin the presence of varying amounts of enzymes or substrates (acyl-CoAintermediates).

FIG. 5 shows the accumulation of pathway intermediates for the C4 to C6elongation cycle observed while varying the enzymes for the ketoacyl-CoA reductase (KCR) and 3-hydroxyacyl-CoA dehydratase (3HCDh)reactions. As shown, when the pathway was reconstructed with FadB, therewas significant accumulation of 3-hydroxybutyryl-CoA, suggestinginsufficient 3HCDh activity to drive the reaction forward. The observedaccumulation may be due to the preference for this enzyme to catalyzethe reverse reaction, which may also be reducing overall forward pathwayflux in vivo. However, when FadB is supplemented with Hbd and/or Crt(alternative KCR and 3HCDh enzymes that have significant activity in theforward direction with C4 intermediates), a reduction in3-hydroxybutryl-CoA accumulation was observed. These data suggested thatthe supplemental activity was sufficient to drive the pathway forward tobutyryl-CoA and that integration of Hbd and Crt into production hostsmay enhance production of longer chain fatty acids.

Synthase Mutant Evaluations

FabH variants were isolated from a 96-well plate-based screen developedto detect beneficial mutations with improved activity on C10-CoA.Following the initial screen of >1000 mutants, positive variants weresequenced and activity on C6-C10-CoA was evaluated. As shown in FIG. 13,three of the purified variants identified were confirmed to haveincreased activity on C6-C10-CoA substrates.

Following positive confirmation, FabH mutants were evaluated in vivo.The following strains were constructed by incorporating the FabHmutations into BXE_198 and BXE_233.

Strains were evaluated in the standard 1 mL screening protocol for FAMEproduction in 20 hours (FIG. 14). As shown below, increased methyllaurate observed with the E37K (strain BXE_271) and D196G/K342E (strainsBXE_273 and BXE_243) variants compared with the control strains (strainsBXE_198 and BXE_233). However, as with the hbd/crt module, it isexpected that these mutations may have a greater impact in productionstrains with improved WES activity.

It has been consistently demonstrated with current production strainsthat the required expression of Aagr FabH to achieve target activitiesin lysate requires a significant fraction of the total protein pool.Furthermore, even with a high level of expression, the FabH activity onC10-CoA is often times at or slightly below the target activity.

TABLE 4d The following strains were constructed by incorporating theFabH mutations into BXE_198 and BXE_233. Strain Parent(s) Plasmid 1Plasmid 2 BXE_198 BX_1018 pET-PpstsIH-Aagr-TS Maqu pACYC_PpstsIHnphT7(SV)-ter- PpstsIH-fadB BXE_233 BX_1018 pET_PpstsIH-Aagr-PtpiA-pACYC-PpstsIH-nphT7(SV)-ter- accDA_PrpiA-accB-accCPpstsIH-fadB-PphoE-Mhyd BXE_271 BX_1018 pET_PpstsIH-Aagr(E37K)-pACYC_PpstsIH nphT7(SV)-ter- PTS-Maqu PpstsIH-fadB BXE_272 BX_1018pET_PpstsIH-Aagr(E51G, pACYC_PpstsIH nphT7(SV)-ter- S357G)-PTS-MaquPpstsIH-fadB BXE_273 BX_1018 pET_PpstsIH-Aagr(D196G, pACYC_PpstsIHnphT7(SV)-ter- E342E)-PTS-Maqu PpstsIH-fadB BXE_243 BX_1018pET_PpstsIH-Aagr(D196G, pACYC_PpstsIH nphT7(SV)-ter-K342E)-Ptpi-accDA_PrpiA- PpstsIH-fadB-PphoE-Mhyd accB-accC BXE_244BX_1018 pET_PpstsIH-Aagr(D196G, pACYC_PpstsIH nphT7(SV)-ter-Q219R)-PtpiA-accDA_PrpiA- PpstsIH-fadB-PphoE-Mhyd accB-accC

Example 2 Optimization of Wax Ester Synthase (WES) Activity for MethylLaurate Production

WES candidates were expressed, purified, and evaluated for solubilityand FAME production in vitro. Based on the results of the initialscreen, 21 WES candidates were chosen for further evaluation includingsubstrate specificity and in vivo FAME production. In vitro assays werecompleted by measuring product formation following the addition ofindividual CoA substrates. As summarized in FIG. 6, a range ofactivities was observed on the various substrates and a number ofcandidates were identified based on high overall activity (Svar, Maqu)or specificity for substrates≥C12-CoA (M360.2, Acip). The sequences ofthe WES used are provided in Table 1 above.

As an orthogonal method for testing in vivo activity, nine of the WEScandidates (Table 4f) shown to be active on lauroyl-CoA in the presenceof methanol were expressed in a fatty acid production host.

TABLE 4f Nine WES candidates shown to be active on lauroyl-CoA in thepresence of methanol were expressed in a fatty acid production hostStrain Host Plasmid 1 Plasmid 2 BXE_003 BX_926 583_pET28b-ΔlacI-1005_pACYC-PpstsIH- PpstsIH-fadBA nphT7-ter-TT_PpstsIH- Abor BXE_013BX_926 583_pET28b-ΔlacI- 1071_pACYC-PpstsIH- PpstsIH-fadBAnphT7-ter-PpstsIH- Gpro BXE_016 BX_926 583_pET28b-ΔlacI-1076_pACYC-PpstsIH- PpstsIH-fadBA nphT7-ter-PpstsIH- Requ BXE_017 BX_926583_pET28b-ΔlacI- 1077_pACYC-PpstsIH- PpstsIH-fadBA nphT7-ter-PpstsIH-LMED BXE_018 BX_926 583_pET28b-ΔlacI- 1078_pACYC-PpstsIH- PpstsIH-fadBAnphT7-ter-PpstsIH- Aazu BXE_019 BX_926 583_pET28b-ΔlacI-1079_pACYC-PpstsIH- PpstsIH-fadBA nphT7-ter-PpstsIH- Msme BXE_020 BX_926583_pET28b-ΔlacI- 1080_pACYC-PpstsIH- PpstsIH-fadBA nphT7-ter-PpstsIH-Ajun BXE_021 BX_926 583_pET28b-ΔlacI- 1081_pACYC-PpstsIH- PpstsIH-fadBAnphT7-ter-PpstsIH- M360.2 BXE_022 BX_926 583_pET28b-ΔlacI-1082_pACYC-PpstsIH- PpstsIH-fadBA nphT7-ter-PpstsIH- ACIP

Small-scale evaluations were completed for each strain cultured atOD₆₀₀=2.0 in a 20 mL capped glass test tube with a working volume of 2mL. Samples were taken at 24 hours by removing 1 mL of the culture forgrowth and glucose measurement and extracting the remaining broth withMTBE prior to analyzing for FAME production by GC-MS. As shown in FIG.7, methyl laurate titers ranging from 0.04-0.5 g/L were observed,indicating modest in vivo WES activity.

Specificity of product formation observed in vivo was significantlydifferent than what would be predicted based upon the in vitro assays.This discrepancy may have been due to the production profile of thethiolase FFA strains, which produced nearly equivalent titers from C6 toC16 FFA. Due to the limitations of screening the wax ester synthasecandidates in the thiolase strain, subsequent in vivo characterizationwas performed in synthase-based strains, which produced C12 FFA and FAMEproducts at higher specificity, rate and titer.

WES High-Throughput Screening

A 96-well plate, Nile red-based assay has been developed forhigh-throughput quantification of FAME production by fluorescence.Mutant libraries were constructed with Maqu WES. The top 20 mutantsidentified in the screen have been isolated, sequenced, cloned intoproduction hosts and evaluated for FAME and FFA production in the 1 mLmethod. While comprehensive data analysis for the 1 mL confirmations ispending, initial results showed significantly improved FAME productionwith at least two WES constructs identified by this method. Asdemonstrated in FIG. 17, both mutations resulted in improved methyllaurate production when compared with the control strain, whilemaintaining a high degree of specificity.

Similarly, mutants of Mhyd were made and the methyl laurate productiondetermined. The results are shown in FIG. 16.

Example 3 Construction and Evaluation of Methyl Laurate ProducingStrains Engineering of Strains for Methyl Laurate Production

Numerous methyl laurate production strains have been constructed,incorporating key pathway modules developed in Examples 1 and 2 aboveand building upon the malonyl-CoA production technology as described inWO2014026162A1 (OPX Biotechnologies Inc., USA). Small-scale evaluationswere completed for each strain cultured at OD₆₀₀=2.0 in a 20 mL cappedglass test tube with a working volume of 1 mL. Samples were taken at 24hours by extracting the entire culture with MTBE and analyzing for FAMEproduction by GC-MS. This 1 mL protocol was instituted in an effort toreduce volatile loss of FAME products seen during shake flaskevaluations.

TABLE 5 methyl laurate production strains used in Example 3 Strain Basestrain Plasmid 1 Plasmid 2 BXE_233 BX_1018 pET_PpstsIH-Aagr-PtpiA-pACYC-PpstsIH-nphT7(SV)-ter- accDA-PrpiA-accB-accCPpstsIH-fadB-PphoE-Mhyd BXE_275 BX_1018 pET_PpstsIH-Aagr-PtpiA-pACYC-PpstsIH-nphT7(SV)-ter- accDA-PrpiA-accB-accCPpstsIH-fadB-PphoE-Aazu BXE_276 BX_1018 pET_PpstsIH-Aagr-PtpiA-pACYC-PpstsIH-nphT7(SV)-ter- accDA-PrpiA-accB-accCPpstsIH-fadB-PphoE-Hche BXE_279 BX_1018 pET_PpstsIH-Aagr-PtpiA-pACYC-PpstsIH-nphT7(SV)-ter- accDA-PrpiA-accB-accCPpstsIH-fadB-PphoE-Maqu

In FIG. 8, 24 hour FAME production data is shown for 50 of the >100methyl laurate producing strains screened in the 1 ml assay since theMay SCM. In this strain set, titers>1 g/L have been obtained in 20 hoursof production with biomass levels ˜1 gDCW/L. Furthermore, specificitiesbetween 70-98% on a FAME basis have been observed.

FIG. 15 shows the 1 mL screening results for strains expressing four WEScandidates shown in Table 5. These results show that varying the WESleads to significant changes in methyl laurate production. Furthermore,Hche showed high levels of production. FIG. 18 also shows that while themajority of the activities (FabH, FadB, Ter) were not significantlydifferent between the two hosts, BXE_233 and BXE_062, the WES activitywas nearly three-fold higher in BXE_233 when compared with BXE_062. Thesignificant differences in methyl laurate production seen with differingWES candidates, and with different expression constructs for the sameWES enzyme, support the hypotheses that WES activity remains limiting incurrent production strains.

Example 4 Fermentation Method Development and Optimization for MethylLaurate Production Initial Development of a 1 L Bioprocess for MethylLaurate Production

Development of a 1 L bioprocess for methyl laurate was carried out tosupport evaluation of production strains under pH-controlled conditionsand at biomass concentrations more representative of commercialproduction.

Several process parameters (Table 6) were explored, including pHsetpoint, methanol feeding, temperature profile and incorporation of asecond phase for increased FAME recovery.

A subset of the data generated is shown in FIG. 9. Overall, the methanolfeeding and control strategy did not appear to have a significant impacton methyl laurate production or overall fermentation performance. Theinclusion of a second phase appeared to be a slight, positive effect onFAME recovery. The incorporation of the 35° C. to 37° C. temperatureshift profile showed a positive effect on production, Based on theseresults, the following baseline process as shown in Table 7 wasestablished and utilized for the subsequent 1 L fermentations andevaluations of methyl laurate producing strains engineered in Example 3.

TABLE 6 Several process parameters used in Example 4 Production MethanolFeed 2^(nd) Temperature Production Condition Profile Phase Profile pHMedium Control 1% (v/v) MeOH N/A 37° C. 7.4 FM12 + 9 mM added at TS;bolus phosphate additions every 8-12 hours to maintain 1% MeC14 1% (v/v)MeOH 50 g/L 37° C. 7.4 FM12 + 9 mM FAME added at TS; bolus C14 phosphateadditions every 8-12 FAME hours to maintain 1% 35 C. to 1% (v/v) MeOHN/A 35° C. →37° C. 7.4 FM12 + 9 mM 37 C. added at TS; bolus phosphateadditions every 8-12 hours to maintain 1% MeOH 0.5% (v/v) MeOH N/A 37°C. 7.4 FM12 + 9 mM Feed added at TS; bolus phosphate additions every8-12 hours to maintain 0.5% pH 7 1% (v/v) MeOH N/A 37° C. 7.4 FM12 + 9mM added at TS; bolus phosphate additions every 8-12 hours to maintain1%

TABLE 7 Baseline process used in fermentation of methyl laurateproducing strains engineered in Example 3 Production Methanol FeedTemperature Production Condition Profile 2^(nd) Phase Profile pH MediumBaseline 1% (v/v) MeOH 50 g/L C14 35° C. →37° C. 7 FM12 + Process addedat TS; FAME 9 mM bolus additions at phosphate every 8-12 hours tomaintain 1%

BXE_276 strain was tested as mentioned above. The average productiontime course for the triplicate BXE_276 was run. Fairly constant methyllaurate production rates were observed over 36.5 hours with an averagetiter of 3 g/L methyl laurate. Interestingly, this strain produced asignificant amount of methyl decanoate (3-5 g/L), resulting insignificantly higher total FAME production than observed with previousstrains (FIG. 19). BXE_276 showed higher specificity for production ofmethyl decanoate than methyl laurate in 1 L, which is in contrast to thehigher specificity demonstrated in 1 mL for methyl laurate production(69%).

REFERENCES

-   Heath et al., Frog. Lipid Res. 40(6):467-97 (2001)-   McCue et al., Nucleic Acids Res., 29(3):774-82 (2001)-   Zhang et al., J. Biol. Chem. 277 (18):15558-65 (2002)-   Chang et al., J. Bacteriol. 154(2):756-62 (1983)-   Abdel-Ahmid et al., Microbiol. 147(6):2001-   Mat-Jan et al., J. Bacteriol. 171(1):342-8-   Bunch et al., Microbiol. 143(1):187-95 (1997)-   Enoyl-Acyl Carrier Protein (fabI) Plays a Determinant Role in    Completing Cycles of Fatty Acid Elongation in Escherichia coli,”    Richard J. Heath and Charles 0. Rock, J. Biol. Chem. 270:44, pp.    26538-26543 (1995),-   Beguin, P and Aubert, J-P (1994) FEMS Microbial. Rev. 13: 25-58-   Ohima, K. et al. (1997) Biotechnol. Genet. Eng. Rev. 14: 365414.-   Antonenkov, V., D. et al. Van Veldhoven, P., P., Waelkens, E., and    Mannaerts, G. P. J. Biol. Chem. 1997, 272:26023-26031.-   Reed, 1981-   Arthur Lesk (2008), Introduction to bioinformatics, 3^(rd) edition,-   Thompson et al., Nucleic Acids Research 22, 4637-4680, 1994,-   Katoh et al., Genome Information, 16(1), 22-33, 2005.

U.S. provisional patent application No. 62/044,621 filed Sep. 2, 2015,is incorporated herein by reference.

Numerous modifications and variations on the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

1.-16. (canceled)
 17. A microbial cell for producing at least one fattyacid ester, wherein the cell comprises: (i) a first genetic mutationthat enables the cell to produce at least one fatty acyl coenzyme A(CoA) intermediate by an increase in the expression of at least oneenzyme selected from the group consisting of acetoacetyl-CoA synthase,ketoacyl-CoA synthase (or elongase), ketoacyl-CoA thiolase, enoyl-CoAreductase, ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydrataserelative to a wild type cell; and (ii) a second genetic mutation thatincreases the activity of at least one wax ester synthase in the cellrelative to the wild type cell, wherein the wax ester synthase hassequence identity of at least 80% to a polypeptide of SEQ ID NO: 1-8,and combinations thereof or to a functional fragment of any of thepolypeptides for catalyzing the conversion of the fatty acyl coenzyme Aintermediate to a fatty acid ester.
 18. The cell according to claim 1,wherein the at least one fatty acyl coenzyme A (CoA) intermediatecomprises a C4 to C10 acyl-CoA substrate.
 19. The cell according toclaim 17, further comprising a genetic mutation that results in adecrease in the expression of acetoacetyl-CoA thiolase relative to thewild type cell.
 20. The cell according to claim 17, wherein the cellcomprises a third genetic mutation that reduces the fatty aciddegradation capacity of the cell relative to the wild type cell.
 21. Thecell according to claim 20, wherein the third genetic mutation resultsin a decrease in the expression of at least one enzyme selected from thegroup consisting of acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase,enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase, each relative to thewild type cell.
 22. The cell according to claim 17, wherein the cell isgenetically modified to increase the expression of β-ketoacyl-ACPsynthase III (fabH) relative to the wild type cell.
 23. The cellaccording to claim 22, wherein the β-ketoacyl-ACP synthase III (fabH)has sequence identity of at least 85% to a polypeptide selected from thegroup consisting of SEQ ID NOs: 24-27 and combinations thereof.
 24. Thecell according to claim 17, wherein the cell is genetically modified toincrease the expression of 3-hydroxyacyl coenzyme A dehydratase (3HCDh)and/or keto acyl-CoA reductase (KCR) relative to the wild type cell. 25.The cell according to claim 24, wherein the 3HCDh is crotonase/enoyl-CoAhydratase (Crt) and the KCR is hydroxybutyric dehydrogenase (Hbd). 26.The cell according to claim 25, wherein the Crt has sequence identity ofat least 85% to a polypeptide as set forth in SEQ ID NO:28 and/or theHbd has sequence identity of at least 85% to a polypeptide of SEQ IDNO:29.
 27. The cell according to claim 17, wherein the fatty acid esteris a fatty acid methyl ester (FAME) and/or a fatty acid ethyl ester(FAEE).
 28. The cell according to claim 17, wherein the fatty acid esteris produced in the presence of at least one exogenous alcohol selectedfrom the group consisting of methanol, ethanol, propanol, butanol, andpentanol.
 29. The cell according to claim 17, wherein the cell is amammalian cell, a plant cell, a yeast cell, a fungi cell, or a bacteriacell.
 30. The cell according to claim 29, wherein the cell is a selectedfrom the group consisting of Pseudomonas putida, Escherichia coli, andSaccharomyces cerevisiae.
 31. A method for producing fatty acid ester,the method comprising: contacting at least one fatty acyl coenzyme A(CoA) intermediate with an isolated wax ester synthase that has sequenceidentity of at least 80% to a polypeptide of SEQ ID NO: 1-8.
 32. Amethod for producing at least one fatty acid ester, the methodcomprising culturing a microbial cell which comprises: (i) a firstgenetic mutation that enables the cell to produce at least one fattyacyl coenzyme A (CoA) intermediate by an increase in the expression ofat least one enzyme selected from the group consisting ofacetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase),ketoacyl-CoA thiolase, enoyl-CoA reductase, ketoacyl-CoA reductase and3-hydroxyacyl-CoA dehydratase relative to a wild type cell; and (ii) asecond genetic mutation that increases the activity of at least one waxester synthase in the cell relative to the wild type cell, wherein thewax ester synthase has sequence identity of at least 80% to apolypeptide of SEQ ID NO: 1-8, and combinations thereof or to afunctional fragment of any of the polypeptides for catalyzing theconversion of the fatty acyl coenzyme A intermediate to a fatty acidester.
 33. The cell according to claim 32, wherein the fatty acid esteris a fatty acid methyl ester (FAME) and/or a fatty acid ethyl ester(FAEE).
 34. The cell according to claim 32, wherein the fatty acid esteris produced in the presence of at least one exogenous alcohol selectedfrom the group consisting of methanol, ethanol, propanol, butanol, andpentanol.